Streptomyces species produce a wide variety of secondary metabolites, including the avermectins, which comprise a series of eight related sixteen-membered macrocyclic lactones with potent anthelmintic and insecticidal activity. The eight distinct but closely related compounds are referred to as A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. The "a" series of compounds refers to the natural avermectin wherein the substituent at the C25 position is (S)sec-butyl, and the "b" series refers to those wherein the substituent at the C25 position is isopropyl. The designations "A" and "B" refer to avermectins wherein the substituent at C5 is methoxy and hydroxy, respectively. The numeral "1" refers to avermectins wherein a double bond is present at the C22, 23 position, and the numeral "2" refers to avermectins having a hydrogen at the C22 position and a hydroxy at the C23 position. Among the related avermectins, the B1 type of avermectin is recognized as having the most effective antiparasitc and pesticidal activity, and is therefore the most commercially desirable avermectin.
The avermectins and their production by aerobic fermentation of strains of S. avermitilis are described, among other places, in U.S. Pat. Nos. 4,310,519 and 4,429,042.
The avermectin (ave) genes, like many genes involved in the production of secondary metabolites and other Streptomyces antibiotics, are found clustered together on the bacterial chromosome. The ave gene cluster for avermectin biosynthesis spans a 95 kb genomic fragment of DNA which includes DNA encoding the avermectin polyketide synthase (PKS) (MacNeil et al., 1992, Gene 115:119-125).
The regulation of antibiotic biosynthesis in Streptomyces is perhaps best characterized in the species Streptomyces coelicolor. Four antibiotics produced by S. coelicolor include actinorhodin (Act), undecylprodigiosin (Red), calcium-dependent antibiotic (CDA), and methylenomycin (Mmy). Each of these antibiotics is encoded by a different cluster of genetically distinct genes. Genes have been identified that are linked to either the Act gene cluster or the Red gene cluster that encode products which specifically regulate the expression of the Act biosynthetic gene cluster or the Red biosynthetic gene cluster, respectively. A number of loci containing genes that globally regulate more than one of the antibiotic biosynthetic gene clusters have also been identified. For example, mutations in two independent loci, absA and absB, have been shown to block the synthesis of all four antibiotics in S. coelicolor (Brian et al., 1996, J. Bact. 178:3221-3231). The absA locus has been cloned and characterized, and its gene products have been shown to be involved in a signal transduction pathway which normally acts as a global negative regulator of antibiotic synthesis in S. coelicolor (Brian et al., 1996, above).
U.S. Pat. No. 5,876,987 to Champness et al. relates to hyperproduction of antibiotic in Streptomyces spp. as a result of interruption of the absA locus.
U.S. Pat. No. 5,707,839 to Denoya, and U.S. Pat. No. 5,728,561 to Denoya et al. relate to DNA sequences encoding branched-chain alpha-ketoacid dehydrogenase complexes of Streptomyces and methods for enhancing the production of novel avermectins.
Understanding the mechanism by which Type I polyketide synthase expression is regulated in S. avermitilis will permit genetic manipulation of the ave genes to increase the production of avermectins.